Our goal is to develop a system based on optical methods for creating and testing neural circuits in vitro. We have developed a multiphoton laser technique (Live Cell Substrate Patterning or LCSP) that allows substrates to be patterned after cells have been plated. The ability to alter substrates dynamically in response to new growth enables us to create well-defined simple (few-cell) circuits. Functional tests of these circuits have been made using conventional electrophysiological methods. We now plan to record changes in activity using calcium imaging. Cells will be loaded with a calcium indicator dye or transfected to express a calcium indicator protein. We will also use light stimulation to activate or silence cells expressing channelrhodopsin-2 or halorhodopsin, respectively. This all-optical approach will allow repeated activation and imaging of cells as will be necessary for studies of slow-changing events during synapse formation. For example, it will be possible to address questions such as when synaptic activity first becomes functional and how synapse formation may be altered in response to changes in activity. It will also provide a means for testing a wide range of cellular and molecular mechanisms that may underlie structure or function of circuits. PUBLIC HEALTH RELEVANCE: New approaches are needed to create and test neural circuits in order to model neural function. Using minimally invasive optical methods we will be able to control circuit formation and also stimulate and record activity at high resolution over time.